Biomedical subjects

F M Hendrickson

Publications and source records attributed to F M Hendrickson.

4 recordsLinked to original sources

PubMed1999

Performance of a 2k CCD camera designed for electron crystallography at 400 kV.

We discuss the performance of a charge-coupled device (CCD) camera that has been designed for use in electron crystallographic studies of proteins. There have been many previous publications describing the characteristics and performance of CCD-based cameras in electron microscopy; here we focus on characteristics relevant to protein studies at 400 kV. The low exposure that must be used in such studies produces a very poor signal-to-noise ratio, so any loss of signal-to-noise ratio in the recording process must be avoided. Images must contain a sufficient number of molecules to allow identification of the reciprocal lattice, thus requiring a large image format. Electron diffraction patterns may contain some spots with intensity around 10(-7) times that of the central beam, so the largest possible dynamic range is helpful. Some of the characteristics we discuss are most easily measured with crystals, but the conclusions also apply for other work such as single-particle analyses. The camera has been optimized for work at 400 kV with a P43 scintillator fiber-optically coupled to a CCD with 24 microns pixels. The scintillator in this camera is thicker than generally used at lower voltages, which provides an adequate signal level but slightly degrades the resolution. Operation at 400 kV leads to a point spread function that is broader than the CCD pixel size. Images are thus binned by a factor of two to double the effective pixel size, with the resulting loss of a factor of two in the size of areas that can be recorded in a single frame. A large CCD with a 2048 x 2048 pixel array is used to compensate for this loss and provide a sufficient signal for the crystallographic image processing used in this work. Images and electron diffraction patterns recorded on the CCD are compared with data recorded on photographic film. While the quality of the images recorded on the CCD at the low exposures required in protein studies is not quite as good as that on film, electron diffraction data recorded on the CCD are superior to that on film.

Crystallography↗

PubMed1998

Structural characterization of the L-to-M transition of the bacteriorhodopsin photocycle.

Structural intermediates occurring in the photocycle of wild-type bacteriorhodopsin are trapped by illuminating hydrated, glucose-embedded purple membrane at 170 K, 220 K, 230 K, and 240 K. We characterize light-induced changes in protein conformation by electron diffraction difference Fourier maps, and relate these to previous work on photocycle intermediates by infrared (FTIR) spectroscopy. Samples illuminated at 170 K are confirmed by FTIR spectroscopy to be in the L state; a difference Fourier projection map shows no structural change within the 0.35-nm resolution limit of our data. Difference maps obtained with samples illuminated at 220 K, 230 K, and 240 K, respectively, reveal a progressively larger structural response in helix F when the protein is still in the M state, as judged by the FTIR spectra. Consistent with previous structural studies, an adjustment in the position or in the degree of ordering of helix G accompanies this motion. The model of the photocycle emerging from this and previous studies is that bacteriorhodopsin experiences minimal change in protein structure until a proton is transferred from the Schiff base to Asp85. The M intermediate then undergoes a conformational evolution that opens a hydrated "half-channel," allowing the subsequent reprotonation of the Schiff base by Asp96.

Aspartic Acid↗

PubMed1994

Selectivity in the interaction of various DNA sequences with H1 histone.

An assay for the binding of H1 histone by DNA was developed based on extraction with phenol, which partitions free DNA into the aqueous layer and aggregates of H1 histone-DNA complexes into the phenol layer and interface. When this assay was performed on fragments of simian virus 40 (SV40) DNA, fragments containing the 21-bp repetitive element and a portion of the origin of replication were resistant to H1 binding. This result was corroborated when an endonuclease protection assay showed that the origin was poorly protected by H1 compared to other sites. DNase I protection mapping demonstrated that H1 "underprotected" sites immediately to either side of the AT element, which lies in the origin of replication. These sites were also hypersensitive to attack by hydroxyl radical in the absence of histone, probably indicative of some conformation aberration such as minor-groove distension. The same DNA sequences resistant to binding H1 histone resisted binding to H4 histone but showed much less selectivity, if any, in binding polylysine. These results clearly demonstrate that the interaction of DNA and H1 (and H4 histone) is more complicated than just charge neutralization and probably involves the conformation of the DNA.

Animals↗

PubMed1985

Defective interfering virus particles modulate virulence.

To determine whether defective interfering (DI) particles modulate virulence by initiating a cyclic pattern of virus growth in vivo, adult mice were infected with vesicular stomatitis virus (VSV), both with and without DI particles. A total of 184 mice divided into groups were inoculated intranasally. A majority of mice inoculated only with standard VSV developed paralysis, most of them between days 7 and 9. The addition of DI particles altered the development of paralysis in several ways. When there was significant protection, a few still became paralyzed on days 7 and 9. When overall mortality was unaffected or even slightly increased, the majority of mice became paralyzed between days 7 and 9 as well. Protection could not be predicted based on a single ratio of standard VSV to DI particles or on the absolute amount of DI particles inoculated. Infectious virus recovered from mouse brains at the time of paralysis and incipient death showed considerable variation, although the titer in a majority of the animals was between 10(5) and 10(7) PFU/ml. When the brains of these paralyzed mice were examined for hybridizable VSV RNA, the detection of standard VSV RNA correlated well with infectivity. The amount of DI RNA in the coinfected mice was more variable and independent of the amount of 40S RNA, although DI RNA was usually found when standard RNA was present. Survivors examined between days 14 and 21 did not contain infectious virus or any detectable viral RNA in their brains. Because these results were consistent with the hypothesis of viral cycling in vivo, rather than a gradual accumulation of total infectious virus, mice were coinfected with 10(8) PFU of standard VSV and 10(5) PFU equivalents of DI particles and sacrificed daily thereafter, irrespective of whether they developed paralysis. Infectivity measurements indicated a reproducible cycling pattern of VSV in the mouse brains with a periodicity of about 5 days. This cycling and the detection of DI RNA in brains several days after intranasal inoculation suggest that there is a dynamic continuous interaction between standard VSV and its DI particle beyond the initial site of replication as the virus population spreads into the host animal. Such cycling of virus production before the full development of specific immune responses from the host may have important implications for viral diagnostics and disease transmission.

Animals↗